Patient deprivation and perceived scan burden negatively impact the quality of whole-body MRI.

AIM: To evaluate the association between the image quality of cancer staging whole-body magnetic resonance imaging (WB-MRI) and patient demographics, distress, and perceived scan burden. MATERIALS AND METHODS: A sample of patients recruited prospectively to multicentre trials comparing WB-MRI with standard scans for staging lung and colorectal cancer were invited to complete two questionnaires. The baseline questionnaire, administered at recruitment, collated data on demographics, distress and co-morbidity. The follow-up questionnaire, completed after staging investigations, measured perceived WB-MRI scan burden (scored 1 low to 7 high). WB-MRI anatomical coverage, and technical quality was graded by a radiographic technician and grading combined to categorise the scan as "optimal", "sub-optimal" or "degraded". A radiologist categorised 30 scans to test interobserver agreement. Data were analysed using the chi-square, Fisher's exact, t-tests, and multinomial regression. RESULTS: One hundred and fourteen patients were included in the study (53 lung, 61 colorectal; average age 65.3 years, SD=11.8; 66 men [57.9%]). Overall, 45.6% (n=52), scans were classified as "optimal" quality, 39.5% (n=45) "sub-optimal", and 14.9% (n=17) as "degraded". In adjusted analyses, greater deprivation level and higher patient-reported scan burden were both associated with a higher likelihood of having a sub-optimal versus an optimal scan (odds ratio [OR]: 4.465, 95% confidence interval [CI]: 1.454 to 13.709, p=0.009; OR: 1.987, CI: 1.153 to 3.425, p=0.013, respectively). None of the variables predicted the likelihood of having a degraded scan. CONCLUSIONS: Deprivation and patients' perceived experience of the WB-MRI are related to image quality. Tailored protocols and individualised patient management before and during WB-MRI may improve image quality.

WNK1/HSN2 founder mutation in patients with hereditary sensory and autonomic neuropathy: A Japanese cohort study.

The clinical and genetic spectrum of hereditary sensory and autonomic neuropathy (HSAN) is still unknown in Japan. We collected a broad cohort of 33 unrelated patients with predominant sensory and/or autonomic dysfunctions, who were referred to our genetic laboratory. A gene panel sequencing targeting 18 HSAN-related genes was performed using a next-generation sequencing system. A recurrent frame shift mutation in the WNK1/HSN2 gene, c.3237_3238insT (p.Asp1080*), was detected in 5 patients. This mutation was homozygous in 4 cases and of a compound heterozygous genotype in 1 case. Geographic and haplotype analysis of all 5 patients suggested a founder event. In addition, a novel heterozygous nonsense variant, c.2615C>G (p.Ser872*), was identified. All the 5 patients presented with severe sensory and autonomic dysfunctions at birth or during adolescence. In 2 patients, an uncommon phenotype of acute pathological pain presented at ~50 years of age. Here, we present the first founder mutation of WNK1/HSN2, in addition to French Canadian, which accounts for ~15.2% of Japanese patients with HSAN in our cohort. We have also reviewed all previously described mutations in WNK1/HSN2 and reconciled their nomenclature strategy on the basis of the current longest transcript.

Down-regulation of the two-component system and cell-wall biosynthesis-related genes was associated with the reversion to daptomycin susceptibility in daptomycin non-susceptible methicillin-resistant Staphylococcus aureus.

Daptomycin (DAP) is widely used in the treatment of methicillin-resistant Staphylococcus aureus (MRSA) infection. The emergence of DAP non-susceptible MRSA strains during therapy is a major concern in clinical settings. Recent studies revealed that MRSA spontaneously reverts to a subsequent methicillin-susceptible S. aureus (MSSA) strain. However, it is not clear whether DAP non-susceptible MRSA has the ability to revert to a susceptible strain. We obtained an MRSA strain pair, DAP non-susceptible strain and subsequent DAP susceptible strain, from a patient. To understand the underlying mechanism by which DAP non-susceptible MRSA reverts to a susceptible strain, we performed genetic and phenotypic analysis in the strain pair. Although whole-genome analysis revealed four missense mutations, including L826F in mprF, in both strains, the net cell-surface charge was similar between the DAP non-susceptible and susceptible strains. However, the thickness of the cell wall was higher in the DAP non-susceptible strain, which was decreased to the same level as the control after reversion to the DAP susceptible strain. Moreover, the non-susceptible strain showed higher mRNA expression of the two-component system (TCS), such as VraSR, yycG and GraS, with the up-regulated transcription levels of cell-wall biosynthesis-related genes. The expression levels of those genes were decreased after reversion to the susceptible strain. These results indicated that DAP non-susceptibility due to up-regulation of the TCS and cell-wall biosynthesis-related genes may be reversible by the discontinuation of DAP, leading to reversion to the DAP susceptible phenotype.

Patient-controlled bolus femoral nerve block after knee arthroplasty: quadriceps recovery, analgesia, local anesthetic consumption.

BACKGROUND: Continuous femoral nerve block (cFNB) induces quadriceps muscle weakness, but patient-controlled femoral nerve block (PCFNB) can provide analgesia with lower consumption of local anesthetics compared to cFNB. We hypothesized that cFNB followed by PCFNB leads to accelerated recovery of quadriceps weakness after total knee arthroplasty compared to cFNB alone. Secondary outcomes were local anesthetic consumption, pain, and mobilization. METHODS: Fifty-six subjects received post-operative cFNB for 24 h and then randomized to receive either cFNB (basal infusion of 6 ml/h using a dummy bolus button; n = 27) or PCFNB (bolus infusion of 3 ml with a lockout time of 30 min and no basal infusion; n = 29) using 0.08% levobupivacaine for the subsequent 24 h in a double-blind manner (registration: UMIN000010105). Quadriceps strength was assessed using a hand-held dynamometer. The percentage change from baseline was compared between groups. RESULTS: Quadriceps strength at 48 h was 47.3 ± 18.3% in the cFNB group and 49.7 ± 15.7% in the PCFNB group (95% confidence interval -7.0-11.9%, P = 0.61). Local anesthetic consumption during the post-operative period was significantly lower in the PCFNB group compared to the cFNB group (102 ± 10.8 ml vs.146 ± 4.6 ml; P < 0.001). No significant differences were found in any of the other outcomes, including pain scores at rest and during knee rehabilitation. CONCLUSION: Continuous femoral nerve block followed by PCFNB does not improve quadriceps strength recovery time compared to cFNB alone after total knee arthroplasty, but similar analgesic effects were demonstrated with reduced levobupivacaine consumption.

A notable case report of May-Hegglin anomaly with immune complex-related nephropathy: a genetic and histological analysis.

May-Hegglin anomaly (MHA) is a rare autosomal dominant disease characterized by macrothrombocytopenia and leukocyte inclusions with microfilaments in the ribosomes. Mutations in the MYH9 gene, encoding non-muscle myosin heavy chain IIA (NMMHC-IIA) have been identified in patients with MHA and other MYH9-related diseases. Two young males (an older and younger brother) presented with macrothrombocytopenia and leukocyte inclusion bodies. Electron microscopy (EM) revealed parallel filaments in leukocyte inclusion bodies characteristic of MHA. Immunofluorescence microscopy (IF) showed NMMHC-IIA antibodies in 1 - 2 leukocyte inclusion bodies. These findings were consistent with MHA and they were identified to express the MYH9 mutation, D1424H. The older brother underwent a renal biopsy because of persistent proteinuria. Histology revealed mesangial proliferative glomerulonephritis with granular deposits of IgG and C1q. EM showed that the dense deposits were located in subendothelial cells, mesangial cells and Bowman's capsule. Immunocytochemistry revealed that NMMHC-IIA antibodies were localized in podocyte and endothelial cells in the glomerulus. Moreover, the expression of nephrin and podocin, slit diagram protein, was normal. An inflammatory mechanism may occur separately from MYH9-related disease. This report presents a case of MHA with immune complex-related nephropathy.

Patient-Relevant Deficits Dictate Endovascular Thrombectomy Decision-Making in Patients with Low NIHSS Scores with Medium-Vessel Occlusion Stroke.

BACKGROUND AND PURPOSE: There is a paucity of evidence regarding the safety of endovascular treatment for patients with acute ischemic stroke due to primary medium-vessel occlusion. The aim of this study was to examine the willingness among stroke physicians to perform endovascular treatment in patients with mild-yet-disabling deficits due to medium-vessel occlusion. MATERIALS AND METHODS: In an international cross-sectional survey consisting of 7 primary medium-vessel occlusion case scenarios, participants were asked whether the presence of personally disabling deficits would influence their decision-making for endovascular treatment despite the patients having low NIHSS scores (<6). Decision rates were calculated on the basis of physician characteristics. Univariable logistic regression clustered by respondent and scenario identity was performed. RESULTS: Three hundred sixty-six participants from 44 countries provided 2562 answers to the 7 medium-vessel occlusion scenarios included in this study. In scenarios in which the deficit was relevant to the patient's profession, 56.9% of respondents opted to perform immediate endovascular treatment compared with 41.0% when no information regarding the patient's profession was provided (risk ratio = 1.39, P < .001). The largest effect sizes were seen for female participants (risk ratio = 1.68; 95% CI, 1.35-2.09), participants older than 60 years of age (risk ratio = 1.61; 95% CI, 1.23-2.10), those with more experience in neurointervention (risk ratio = 1.60; 95% CI, 1.24-2.06), and those who personally performed >100 endovascular treatments per year (risk ratio = 1.63; 95% CI, 1.22-2.17). CONCLUSIONS: The presence of a patient-relevant deficit in low-NIHSS acute ischemic stroke due to medium-vessel occlusion is an important factor for endovascular treatment decision-making. This may have relevance for the conduct and interpretation of low-NIHSS endovascular treatment in randomized trials.

Neck Location on the Outer Convexity is a Predictor of Incomplete Occlusion in Treatment with the Pipeline Embolization Device: Clinical and Angiographic Outcomes.

BACKGROUND AND PURPOSE: With the increasing use of the Pipeline Embolization Device for the treatment of aneurysms, predictors of clinical and angiographic outcomes are needed. This study aimed to identify predictors of incomplete occlusion at last angiographic follow-up. MATERIALS AND METHODS: In our retrospective, single-center cohort study, 105 ICA aneurysms in 89 subjects were treated with Pipeline Embolization Devices. Patients were followed per standardized protocol. Clinical and angiographic outcomes were analyzed. We introduced a new morphologic classification based on the included angle of the parent artery against the neck location: outer convexity type (included angle, <160°), inner convexity type (included angle, >200°), and lateral wall type (160° ≤ included angle ≤200°). This classification reflects the metal coverage rate and flow dynamics. RESULTS: Imaging data were acquired in 95.3% of aneurysms persistent at 6 months. Complete occlusion was achieved in 70.5%, and incomplete occlusion, in 29.5% at last follow-up. Multivariable regression analysis revealed that 60 years of age or older (OR, 5.70; P = .001), aneurysms with the branching artery from the dome (OR, 10.56; P = .002), fusiform aneurysms (OR, 10.2; P = .009), and outer convexity-type saccular aneurysms (versus inner convexity type: OR, 30.3; P < .001; versus lateral wall type: OR, 9.71; P = .001) were independently associated with a higher rate of incomplete occlusion at the last follow-up. No permanent neurologic deficits or rupture were observed in the follow-up period. CONCLUSIONS: The aneurysm neck located on the outer convexity is a new, incomplete occlusion predictor, joining older age, fusiform aneurysms, and aneurysms with the branching artery from the dome. No permanent neurologic deficits or rupture was observed in the follow-up, even with incomplete occlusion.

Perceived Limits of Endovascular Treatment for Secondary Medium-Vessel-Occlusion Stroke.

BACKGROUND AND PURPOSE: Thrombus embolization during mechanical thrombectomy occurs in up to 9% of cases, making secondary medium vessel occlusions of particular interest to neurointerventionalists. We sought to gain insight into the current endovascular treatment approaches for secondary medium vessel occlusion stroke in an international case-based survey because there are currently no clear recommendations for endovascular treatment in these patients. MATERIALS AND METHODS: Survey participants were presented with 3 cases involving secondary medium vessel occlusions, each consisting of 3 case vignettes with changes in the patient's neurologic status (improvement, no change, unable to assess). Multivariable logistic regression analyses clustered by the respondent's identity were used to assess factors influencing the decision to treat. RESULTS: In total, 366 physicians (56 women, 308 men, 2 undisclosed) from 44 countries provided 3294 responses to 9 scenarios. Most (54.1%, 1782/3294) were in favor of endovascular treatment. Participants were more likely to treat occlusions in the anterior M2/3 (74.3%; risk ratio = 2.62; 95% CI, 2.27-3.03) or A3 (59.7%; risk ratio = 2.11; 95% CI, 1.83-2.42) segment compared with the M3/4 segment (28.3%; reference). Physicians were less likely to pursue endovascular treatment in patients who showed neurologic improvement than in patients with an unchanged neurologic deficit (49.9% versus 57.0% responses in favor of endovascular treatment, respectively; risk ratio = 0.88, 95% CI, 0.83-0.92). Interventionalists and more experienced physicians were more likely to treat secondary medium vessel occlusions. CONCLUSIONS: Physicians' willingness to treat secondary medium vessel occlusions endovascularly is limited and varies per occlusion location and change in neurologic status. More evidence on the safety and efficacy of endovascular treatment for secondary medium vessel occlusion stroke is needed.

Two patients with focal segmental glomerulosclerosis complicated by cyclosporine-induced reversible posterior leukoencephalopathy syndrome.

Reversible posterior leukoencephalopathy syndrome (RPLS) is a distinctive clinicoradiological entity observed in a variety of clinical settings. Cyclosporine (CyA)-RPLS has been reported in a few patients with focal segmental glomerulosclerosis (FSGS); however, there had been no reports on developed RPLS after the re-administration of CyA treatment. We report two patients with FSGS who developed CyA-induced RPLS and summarize the results of a literature review for similar patients. The two patients with FSGS presented here were a 4-year-old boy and a 9-year-old boy, who presented with steroid-resistant nephrotic syndrome (NS) and were treated with CyA. The first patient developed CyA-induced RPLS at the 7th day after the start of CyA treatment, and the second patient at the 16th day after the re-start of CyA treatment. The two patients complained of a visual disorder and exhibited signs of a disturbance in consciousness and hypertension. Electroencephalography (EEG) examinations revealed a generalized slow wave pattern, and magnetic resonance imaging (MRI) disclosed an area of high signal intensity in the white matter. Subsequently, CyA was discontinued and neurological symptoms improved and recrudescence of RPLS did not occur. Our findings suggest that patients with FSGS and NS who are treated with CyA should be closely monitored for the possible onset of RPLS, presenting as a disturbance in consciousness, visual disturbances and/or convulsions.

Complications of Endovascular Treatments for Brain Arteriovenous Malformations: A Nationwide Surveillance.

BACKGROUND AND PURPOSE: Embolization is widely performed to treat brain arteriovenous malformations, but little has been reported on factors contributing to complications. We retrospectively reviewed a nationwide surveillance to identify risk factors contributing to complications and short-term clinical outcomes in the endovascular treatment of brain arteriovenous malformations. MATERIALS AND METHODS: Data for endovascular treatment of brain arteriovenous malformations were extracted from the Japanese nationwide surveillance. Patient characteristics, brain arteriovenous malformation features, procedures, angiographic results, complications, and clinical outcomes at 30 days postprocedure were analyzed. RESULTS: A total of 1042 endovascular procedures (788 patients; mean, 1.43 ± 0.85 procedures per patient) performed in 111 institutions from 2010 to 2014 were reviewed. Liquid materials were used in 976 procedures (93.7%): to perform presurgical embolization in 638 procedures (61.2%), preradiosurgical embolization in 160 (15.4%), and as sole endovascular treatment in 231 (22.2%). Complete or near-complete obliteration of brain arteriovenous malformations was obtained in 386 procedures (37.0%). Procedure-related complications occurred in 136 procedures (13.1%), including hemorrhagic complications in 59 (5.7%) and ischemic complications in 57 (5.5%). Univariate analysis identified deep venous drainage, associated aneurysms, infratentorial location, and preradiosurgical embolization as statistically significant risk factors for complications. Multivariate analysis showed that embolization of brain arteriovenous malformations in the infratentorial location was significantly associated with complications. Patients with complications due to endovascular procedures had worse clinical outcomes 30 days after the procedures than those without complications. CONCLUSIONS: Complications arising after endovascular treatment of brain arteriovenous malformations are not negligible even though they may play a role in adjunctive therapy, especially in the management of infratentorial brain arteriovenous malformations.

Predictors of Cerebral Aneurysm Rupture after Coil Embolization: Single-Center Experience with Recanalized Aneurysms.

BACKGROUND AND PURPOSE: Recanalization after coil embolization is widely studied. However, there are limited data on how recanalized aneurysms rupture. Herein, we describe our experience with the rupture of recanalized aneurysms and discuss the type of recanalized aneurysms at greatest rupture risk. MATERIALS AND METHODS: A total of 426 unruptured aneurysms and 169 ruptured aneurysms underwent coil embolization in our institution between January 2009 and December 2017. Recanalization occurred in 38 (8.9%) of 426 unruptured aneurysms (unruptured group) and 37 (21.9%) of 169 ruptured aneurysms (ruptured group). The Modified Raymond-Roy classification on DSA was used to categorize the recanalization type. Follow-up DSA was scheduled until 6 months after treatment, and follow-up MRA was scheduled yearly. If recanalization was suspected on MRA, DSA was performed. RESULTS: In the unruptured group, the median follow-up term was 74.0 months. Retreatment for recanalization was performed in 18 aneurysms. Four of 20 untreated recanalized aneurysms (0.94% of total coiled aneurysms) ruptured. In untreated recanalized aneurysms, class IIIb aneurysms ruptured significantly more frequently than class II and IIIa (P = .025). In the ruptured group, the median follow-up term was 28.0 months. Retreatment for recanalization was performed in 16 aneurysms. Four of 21 untreated recanalized aneurysms (2.37% of total coiled aneurysms) ruptured. Class IIIb aneurysms ruptured significantly more frequently than class II and IIIa (P = .02). CONCLUSIONS: The types of recanalization after coil embolization may be predictors of rupture. Coiled aneurysms with class IIIb recanalization should undergo early retreatment because of an increased rupture risk.

Early detection of Niemann-pick disease type C with cataplexy and orexin levels: continuous observation with and without Miglustat.

STUDY OBJECTIVES: Niemann-Pick type C (NPC) is an autosomal recessive and congenital neurological disorder characterized by the accumulation of cholesterol and glycosphingolipids. Symptoms include hepatosplenomegaly, vertical supranuclear saccadic palsy, ataxia, dystonia, and dementia. Some cases frequently display narcolepsy-like symptoms, including cataplexy which was reported in 26% of all NPC patients and was more often recorded among late-infantile onset (50%) and juvenile onset (38%) patients. In this current study, we examined CSF orexin levels in the 10 patients of NPC with and without cataplexy, which supports previous findings. METHODS: Ten patients with NPC were included in the study (5 males and 5 females). NPC diagnosis was biochemically confirmed in all 10 patients, from which 8 patients with NPC1 gene were identified. We compared CSF orexin levels among NPC, narcoleptic and idiopathic hypersomnia patients. RESULTS: Six NPC patients with cataplexy had low or intermediate orexin levels. In 4 cases without cataplexy, their orexin levels were normal. In 5 cases with Miglustat treatment, their symptoms stabilized or improved. For cases without Miglustat treatment, their conditions worsened generally. The CSF orexin levels of NPC patients were significantly higher than those of patients with narcolepsy-cataplexy and lower than those of patients with idiopathic hypersomnia, which was considered as the control group with normal CSF orexin levels. DISCUSSION: Our study indicates that orexin level measurements can be an early alert of potential NPC. Low or intermediate orexin levels could further decrease due to reduction in the neuronal function in the orexin system, accelerating the patients' NPC pathophysiology. However with Miglustat treatment, the orexin levels stabilized or improved, along with other general symptoms. Although the circuitry is unclear, this supports that orexin system is indeed involved in narcolepsy-cataplexy in NPC patients. CONCLUSION: The NPC patients with cataplexy had low or intermediate orexin levels. In the cases without cataplexy, their orexin levels were normal. Our study suggests that orexin measurements can serve as an early alert for potential NPC; furthermore, they could be a marker of therapy monitoring during a treatment.

Antiplatelet Management for Stent-Assisted Coiling and Flow Diversion of Ruptured Intracranial Aneurysms: A DELPHI Consensus Statement.

BACKGROUND AND PURPOSE: There is a paucity of data regarding antiplatelet management strategies in the setting of stent-assisted coiling/flow diversion for ruptured intracranial aneurysms. This study aimed to identify current challenges in antiplatelet management during stent-assisted coiling/flow diversion for ruptured intracranial aneurysms and to outline possible antiplatelet management strategies. MATERIALS AND METHODS: The modified DELPHI approach with an on-line questionnaire was sent in several iterations to an international, multidisciplinary panel of 15 neurointerventionalists. The first round consisted of open-ended questions, followed by closed-ended questions in the subsequent rounds. Responses were analyzed in an anonymous fashion and summarized in the final manuscript draft. The statement received endorsement from the World Federation of Interventional and Therapeutic Neuroradiology, the Japanese Society for Neuroendovascular Therapy, and the Chinese Neurosurgical Society. RESULTS: Data were collected from December 9, 2019, to March 13, 2020. Panel members achieved consensus that platelet function testing may not be necessary and that antiplatelet management for stent-assisted coiling and flow diversion of ruptured intracranial aneurysms can follow the same principles. Preprocedural placement of a ventricular drain was thought to be beneficial in cases with a high risk of hydrocephalus. A periprocedural dual, intravenous, antiplatelet regimen with aspirin and a glycoprotein IIb/IIIa inhibitor was preferred as a standard approach. The panel agreed that intravenous medication can be converted to oral aspirin and an oral P2Y12 inhibitor within 24 hours after the procedure. CONCLUSIONS: More and better data on antiplatelet management of patients with ruptured intracranial aneurysms undergoing stent-assisted coiling or flow diversion are urgently needed. Panel members in this DELPHI consensus study preferred a periprocedural dual-antiplatelet regimen with aspirin and a glycoprotein IIb/IIIa inhibitor.

Considerations for Antiplatelet Management of Carotid Stenting in the Setting of Mechanical Thrombectomy: A Delphi Consensus Statement.

BACKGROUND AND PURPOSE: There are only few data and lack of consensus regarding antiplatelet management for carotid stent placement in the setting of endovascular stroke treatment. We aimed to develop a consensus-based algorithm for antiplatelet management in acute ischemic stroke patients undergoing endovascular treatment and simultaneous emergent carotid stent placement. MATERIALS AND METHODS: We performed a literature search and a modified Delphi approach used Web-based questionnaires that were sent in several iterations to an international multidisciplinary panel of 19 neurointerventionalists from 7 countries. The first round included open-ended questions and formed the basis for subsequent rounds, in which closed-ended questions were used. Participants continuously received feedback on the results from previous rounds. Consensus was defined as agreement of ≥70% for binary questions and agreement of ≥50% for questions with >2 answer options. The results of the Delphi process were then summarized in a draft manuscript that was circulated among the panel members for feedback. RESULTS: A total of 5 Delphi rounds were performed. Panel members preferred a single intravenous aspirin bolus or, in jurisdictions in which intravenous aspirin is not available, a glycoprotein IIb/IIIa receptor inhibitor as intraprocedural antiplatelet regimen and a combination therapy of oral aspirin and a P2Y12 inhibitor in the postprocedural period. There was no consensus on the role of platelet function testing in the postprocedural period. CONCLUSIONS: More and better data on antiplatelet management for carotid stent placement in the setting of endovascular treatment are urgently needed. Panel members preferred intravenous aspirin or, alternatively, a glycoprotein IIb/IIIa receptor inhibitor as an intraprocedural antiplatelet agent, followed by a dual oral regimen of aspirin and a P2Y12 inhibitor in the postprocedural period.

Distinct effects of fatty acids on translocation of gamma- and epsilon-subspecies of protein kinase C.

Effects of fatty acids on translocation of the gamma- and epsilon-subspecies of protein kinase C (PKC) in living cells were investigated using their proteins fused with green fluorescent protein (GFP). gamma-PKC-GFP and epsilon-PKC-GFP predominated in the cytoplasm, but only a small amount of gamma-PKC-GFP was found in the nucleus. Except at a high concentration of linoleic acid, all the fatty acids examined induced the translocation of gamma-PKC-GFP from the cytoplasm to the plasma membrane within 30 s with a return to the cytoplasm in 3 min, but they had no effect on gamma-PKC-GFP in the nucleus. Arachidonic and linoleic acids induced slow translocation of epsilon-PKC-GFP from the cytoplasm to the perinuclear region, whereas the other fatty acids (except for palmitic acid) induced rapid translocation to the plasma membrane. The target site of the slower translocation of epsilon-PKC-GFP by arachidonic acid was identified as the Golgi network. The critical concentration of fatty acid that induced translocation varied among the 11 fatty acids tested. In general, a higher concentration was required to induce the translocation of epsilon-PKC-GFP than that of gamma-PKC-GFP, the exceptions being tridecanoic acid, linoleic acid, and arachidonic acid. Furthermore, arachidonic acid and the diacylglycerol analogue (DiC8) had synergistic effects on the translocation of gamma-PKC-GFP. Simultaneous application of arachidonic acid (25 MicroM) and DiC8 (10 microM) elicited a slow, irreversible translocation of gamma-PKC- GFP from the cytoplasm to the plasma membrane after rapid, reversible translocation, but a single application of arachidonic acid or DiC8 at the same concentration induced no translocation. These findings confirm the involvement of fatty acids in the translocation of gamma- and epsilon-PKC, and they also indicate that each subspecies has a specific targeting mechanism that depends on the extracellular signals and that a combination of intracellular activators alters the target site of PKCs.

Direct visualization of the translocation of the gamma-subspecies of protein kinase C in living cells using fusion proteins with green fluorescent protein.

We expressed the gamma-subspecies of protein kinase C (gamma-PKC) fused with green fluorescent protein (GFP) in various cell lines and observed the movement of this fusion protein in living cells under a confocal laser scanning fluorescent microscope. gamma-PKC-GFP fusion protein had enzymological properties very similar to that of native gamma-PKC. The fluorescence of gamma-PKC- GFP was observed throughout the cytoplasm in transiently transfected COS-7 cells. Stimulation by an active phorbol ester (12-O-tetradecanoylphorbol 13-acetate [TPA]) but not by an inactive phorbol ester (4alpha-phorbol 12, 13-didecanoate) induced a significant translocation of gamma-PKC-GFP from cytoplasm to the plasma membrane. A23187, a Ca2+ ionophore, induced a more rapid translocation of gamma-PKC-GFP than TPA. The A23187-induced translocation was abolished by elimination of extracellular and intracellular Ca2+. TPA- induced translocation of gamma-PKC-GFP was unidirected, while Ca2+ ionophore-induced translocation was reversible; that is, gamma-PKC-GFP translocated to the membrane returned to the cytosol and finally accumulated as patchy dots on the plasma membrane. To investigate the significance of C1 and C2 domains of gamma-PKC in translocation, we expressed mutant gamma-PKC-GFP fusion protein in which the two cysteine rich regions in the C1 region were disrupted (designated as BS 238) or the C2 region was deleted (BS 239). BS 238 mutant was translocated by Ca2+ ionophore but not by TPA. In contrast, BS 239 mutant was translocated by TPA but not by Ca2+ ionophore. To examine the translocation of gamma-PKC-GFP under physiological conditions, we expressed it in NG-108 cells, N-methyl-D-aspartate (NMDA) receptor-transfected COS-7 cells, or CHO cells expressing metabotropic glutamate receptor 1 (CHO/mGluR1 cells). In NG-108 cells , K+ depolarization induced rapid translocation of gamma-PKC-GFP. In NMDA receptor-transfected COS-7 cells, application of NMDA plus glycine also translocated gamma-PKC-GFP. Furthermore, rapid translocation and sequential retranslocation of gamma-PKC-GFP were observed in CHO/ mGluR1 cells on stimulation with the receptor. Neither cytochalasin D nor colchicine affected the translocation of gamma-PKC-GFP, indicating that translocation of gamma-PKC was independent of actin and microtubule. gamma-PKC-GFP fusion protein is a useful tool for investigating the molecular mechanism of gamma-PKC translocation and the role of gamma-PKC in the central nervous system.